Contraction of rabbit skinned skeletal muscle fibers at low levels of magnesium adenosine triphosphate

The contractile properties of skinned single fibers from rabbit psoas muscle were investigated under conditions of low MgATP and no Ca2+ (i.e., less than 10(-8) M). At 1 microM MgATP, fibers shortened at a maximum velocity of 660 +/- 420 A/half sarcomere/s (n = 9), compared with 34,000 A/half sarcomere/s measured during maximum Ca2+-activation at 1 mM MgATP (Moss, R. L., 1982. J. Muscle Res. Cell. Motil ., 3:295-311). The observed dependence of Vmax on pMgATP between 7.0 and 5.3 was similar to that of actomyosin ATPase measured previously by Weber, A., R. Herz , and I. Reiss (1969, Biochemistry, 8:2266-2270). Isometric tension was found to vary with pMgATP in a manner much like that reported by Reuben , J. P., P. W. Brandt, M. Berman , and H. Grundfest (J. Gen. Physiol. 1971. 57:385-407). A simple cross-bridge model was developed to simulate contractile behaviour at both high and low levels of MgATP. It was found that the pMgATP dependence of Vmax and ATPase could be successfully modeled if the rate of detachment of the cross-bridge was made proportional to the concentration of MgATP. In the model, the similar dependence of Vmax and ATPase on pMgATP was derived from the fact that in this range of pMgATP every pass of a cross-bridge by an actin site resulted in an attachment-detachment cycle, and every such cycle caused hydrolysis of one molecule of ATP.     

Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration

The influence of MgATP on the Ga⁺⁺-activated isometric tension of skinned frog muscle fibers was examined in solutions containing: Mg⁺⁺ = 5 mM, creatine phosphate (CP) = 14.5 mM, creatinephosphokinase (CPK) = 1 mg/ml, total EGTA = 7 mM, CaCl₂, KCl, imidazole ≥ 20 mM so that ionic strength = 0.15, pH = 7.00, and MgATP = 2 mM, 0.1 mM, or 20 µM. CP and CPK were necessary for these experiments as determined experimentally by their effect on the tension-Ca⁺⁺ relation, which was saturated for CP ≥ 14.5 mM. This was interpreted to mean that sufficient CP was present to effectively buffer MgATP intracellularly. Decreasing MgATP shifts the tension-pCa curve to higher pCa (-log Ca⁺⁺) so that, for half-maximal tension: pCa_1/2 = 4.5 for MgATP = 2 mM, pCa_1/2 = 5.1 for MgATP = 0.1 mM, and pCa_1/2 = 5.8 for MgATP = 20 µM; maximum isometric tension is the same in all cases, however. If MgATP was decreased to 1 µM, tension at Ga⁺⁺ > 10^–8 M was 84% of the maximum Ca^-+-activated tension in 2 mM MgATP and increased only slightly to 90% for pCa = 4.5. Weber (1970, In The Physiology and Biochemistry of Muscle as Food, Volume 2, E. J. Briskey, R. G. Cassens, and B. B. Marsh, University of Wisconsin Press, Madison, Wis.), using similar solutions, observed similar shifts in half-maximal calcium activation of rabbit myofibril ATPase rates. In explanation, Weber and Bremel (1971, In Contractility of Muscle Cells and Related Processes, R. J. Podolsky, editor, Prentice-Hall, Inc., Englewood Cliffs, N.J.; Bremel and Weber, 1972, Nat. New Biol., 238:97) have described a mechanism whereby, at low ATP, "rigor complexes" are formed between myosin and thin filament actin and, in turn, alter the calcium affinity of one class of the two Ca⁺⁺-binding sites on troponin, so that the thin filament is "turned on" for contraction at lower Ca⁺⁺ levels. Tension data from skinned fibers substantially supports this hypothesis. A stability constant for CaEGTA of 2.62 x 10¹⁰ M^–1 was determined, with the help of F. N. Briggs, in solutions similar to those used for skinned fibers and was the same for 100 and 300 mM KCl.     

Force generation and phosphate release steps in skinned rabbit soleus slow-twitch muscle fibers.

The force-generation and phosphate-release steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibers (STF) were investigated using sinusoidal analysis, and the results were compared with those of rabbit psoas fast-twitch fibers (FTF). Single fiber preparations were activated at pCa 4.40 and ionic strength 180 mM at 20 degrees C. The effects of inorganic phosphate (Pi) concentrations on three exponential processes, B, C, and D, were studied. Results are consistent with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and P is inorganic phosphate. The values determined are k4 = 5.7 +/- 0.5 s-1 (rate constant of isomerization step, N = 9, mean +/- SE), k-4 = 4.5 +/- 0.5 s-1 (rate constant of reverse isomerization), K4 = 1.37 +/- 0.13 (equilibrium constant of the isomerization), and K5 = 0.18 +/- 0.01 mM-1 (Pi association constant). The isomerization step (k4) in soleus STF is 20 times slower, and its reversal (k-4) is 20 times slower than psoas fibers. Consequently, the equilibrium constant of the isomerization step (K4) is the same in these two types of fibers. The Pi association constant (K5) is slightly higher in STF than in FTF, indicating that Pi binds to cross-bridges slightly more tightly in STF than FTF. By correlating the cross-bridge distribution with isometric tension, it was confirmed that force is generated during the isomerization (step 4) of the AMDP state and before Pi release in soleus STF.     

Effect of joule temperature jump on tension and stiffness of skinned rabbit muscle fibers.

The effects of a temperature jump (T-jump) from 5-7 degrees C to 26-33 degrees C were studied on tension and stiffness of glycerol-extracted fibers from rabbit psoas muscle in rigor and during maximal Ca2+ activation. The T-jump was initiated by passing an alternating current pulse (30 kHz, up to 2.5 kV, duration 0.2 ms) through a fiber suspended in air. In rigor the T-jump induces a drop of both tension and stiffness. During maximal activation, the immediate stiffness dropped by (4.4 +/- 1.6) x 10(-3)/1 degree C (mean + SD) in response to the T-jump, and this was followed by a monoexponential stiffness rise by a factor of 1.59 +/- 0.14 with a rate constant ks = 174 +/- 42 s-1 (mean +/- SD, n = 8). The data show that the fiber stiffness, determined by the cross-bridge elasticity, in both rigor and maximal activation is not rubber-like. In the activated fibers the T-jump induced a biexponential tension rise by a factor of 3.45 +/- 0.76 (mean +/- SD, n = 8) with the rate constants 500-1,000 s-1 for the first exponent and 167 +/- 39 s-1 (mean +/- SD, n = 8) for the second exponent. The data are in accordance with the assumption that the first phase of the tension transient after the T-jump is due to a force-generating step in the attached cross-bridges, whereas the second one is related to detachment and reattachment of cross-bridges.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.     

Influence of magnesium on chloride-induced calcium release in skinned muscle fibers

Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization.     

Approximating the isometric force-calcium relation of intact frog muscle using skinned fibers.

In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibrils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested predictions of this calculation using measurements of the potential across the boundary of skinned frog muscle fibers bathed in this solution. The average potential was -3.1 mV, close to that predicted from a simple Donnan equilibrium. The contribution of ATP hydrolysis to a diffusion potential was probably small because addition of 1 mM vanadate to the solution decreased the fiber actomyosin ATPase rate (measured by high-performance liquid chromatography) by at least 73% but had little effect on the measured potential. Using these solutions, we obtained force-pCa curves from mechanically skinned fibers at three different temperatures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temperature. The bath concentration of Ca2+ required for half-maximal activation of isometric force was 1.45 microM (22 degrees C, pH 7.18), 2.58 microM (16 degrees C, pH 7.25), and 3.36 microM (5 degrees C, pH 7.59). The [Ca2+] at the threshold of activation at 16 degrees C was approximately 1 microM, in good agreement with estimates of threshold [Ca2+] in intact frog muscle fibers.     

Effect of dipyridamole on inosine triphosphate pyrophosphohydrolase activity and inosine triphosphate content in fresh human erythrocytes incubated with adenosine

The activity of inosine triphosphate pyrophosphohydrolase (ITPH) in human erythrocytes was found to be 1.50 +/- 0.39 mumol of inosine triphosphate (ITP) hydrolysed x min-1 per g Hb, and no measurable amount of ITP was detected. When dipyridamole was added to the medium composed of adenosine, pyruvate and inorganic phosphate, ITPH activity was 1.18 +/- 0.41, and at the same time ITP accumulation was 0.61 +/- 0.31 mumol/g Hb. The negative correlation between ITPH activity and accumulation of ITP was r = -0.87 at P less than 0.001.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.     

Effect of adenosine triphosphate analogues on skeletal muscle fibers in rigor.

It is commonly believed, for both vertebrate striated and insect flight muscle, that when the ATP analogue adenyl-5'-yl imidodiphosphate (AMPPNP) is added to the muscle fiber in rigor, it causes the fiber to lengthen by 0.15%. This has been interpretated (Marston S.B., C.D. Roger, and R.T. Tregear. 1976. J. Mol. Biol. 104:263-267) as suggesting (a) that in rigor the crossbridge is fixed to, i.e., almost never detaches from the actin filament; (b), that the crossbridge remains fixed to the actin filament after AMPPNP addition; and (c) that the ability of AMPPNP to cause apparent lengthening of a muscle fiber is due to its ability to cause a conformational change in the myosin crossbridge that has an axial component of approximately 1.6 nm/half-sarcomere. The present study, done only on chemically-skinned rabbit psoas fibers, confirms that AMPPNP can cause muscle fibers to lengthen by 0.15% but only for a narrow set of experimental conditions. When experimental conditions are varied over a wider range, it becomes apparent that the extent of lengthening of a rigor muscle fiber upon AMPPNP addition depends almost entirely on the strain present in the rigor fiber before AMPPNP addition. Addition of AMPPNP to an unstrained rigor fiber (one supporting zero tension), induces zero length change while addition of AMPPNP to very highly strained rigor fibers induces length changes greater than 0.15%. The data thus do not support the hypotheses that the crossbridges remain fixed to the actin filament after AMPPNP addition and that the size of the apparent length change induced by AMPPNP is related to the size of the axial component of a conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.